1. Field of the Invention
The present disclosure relates to novel iturin biosynthesis genes, and more particularly, to novel iturin biosynthesis genes derived from Bacillus subtilis subsp. krictiensis ATCC 55079 and uses thereof.
2. Description of the Related Art
Biological control is a means of controlling pathogenic microorganisms that cause disease injuries in plants through the use of other microorganisms that have antagonistic actions. Most representative biopesticides have been primarily used for controlling various plant pathogens, insects harming crops, insect pests such as mites, nematodes, and weeds through direct or indirect use of microorganisms themselves. Studies on this biological control started in early 1990s, and since then, many efforts have been made to inhibit plant pathogens by using various kinds of bacteria and fungi. However, since the soil ecosystems are complex and complicated interactions are associated between plants and microorganisms, the results of studies were very unsatisfying. Recently, interactions between plants and microorganisms have been gradually identified and outstanding results have been reported thanks to developments in biotechnology, such as molecular biology. Currently, about 40 kinds of biological control agents have been developed all around the world (H. D. Burges, Formulation of microbial biopesticides, Kluwer Academic Publisher, Dordrecht, The Netherlands, p. 187-202, 1998) and 25 kinds of biological control agents are registered and commercially available only in the U.S. (B. B. McSpadden Gardener, et al., Plant Health Progress [Internet], May 10, 2002 [cited Aug. 13, 2010].
Registered products are mainly for the control of soil borne plant diseases by Fusarium, Pythium, Rhizoctonia, and Sclerotinia (Phytophthora), but, these products have not yet captured a large share of the market. So far, one of the most successful examples of biological control is the control of crown gall, a disease of roots in fruit trees, caused by Agrobacterium tumefaciens (A. Kerr, Plant Dis., 64: 25-30, 1980). Controlling this plant disease cannot be carried out with chemosynthetic pesticides. However, it has been known that an antibiotic agrocin produced by Agrobacterium radiobacter inhibits the invasion of A. tumefaciens, and then, several products that improved A. radiobacter by using genetic engineering techniques have been developed and thought to have a considerable worldwide market share (names of products: Nogall, Norbac, Galltrol-A, etc.). Another successful example is the study on the control of root rots of wheat using Pseudomonas fluorescens, and research teams at the USDA and Washington State University succeeded in isolating P. fluorescens which exhibits strong antagonistic activity against Gaeumannomyces graminis var. tritici causing the root rots of wheat from soil through 10-year researches (D. M. Weller, Annu. Rev. Phytopathol., 26: 379-407, 1988; D. M. Weller, et al., Can. J. Plant Pathol., 8: 328-334, 1986; R. J. Cook, Can. J. Plant. Pathol., 14: 76-85, 1992). When wheat seeds were treated with this antagonistic microorganism and sown, the yield of wheat increased by 10 to 20% and it was concluded that this effect was caused by phenazine antibiotics (L. S. Thomashow, et al., Appl. Environ. Microbiol., 56: 908-912, 1990; C. Keel, et al., Mol. Plant-Microbe Interact., 5: 4-13, 1992) and 2,4-diacetyl phloroglucinol antibiotic produced by Pseudomonas (C. Keel, et al., Mol. Plant-Microbe Interact., 5: 4-13, 1992). To overcome the differences in control activities depending on application time and region, revealed through a field experiment over 8 years, research teams introduced genetic engineering techniques to maximize the gene expression of Pseudomonas sp. related with the production of phenazine antibiotics and succeeded in overcoming the irregularity of the control effect of root rots of wheat (M. H. Ryder, et al., Improving plant productivity with Rhizobacteria, CSIRO Divisions of soils, Adelaide, South Australia, p. 247-249, 1994).
Bacillus subtilis (B. subtilis) has been received attention of many researchers due to not only many kinds of antibiotics but also its characteristic to produce various enzymes, and along with Saccharomyces cerevisiae (S. cerevisiae), and Lactobacillus sp., it is recognized as a harmless strain to a human body and the environment by the U.S. Food and Drug Administration. Examples of formulations of microbial pesticides developed by using B. subtilis include Epic, Kodiak, Companion, HiStick, Serenade, etc. and these are largely widely used for seed treatment or post-harvest application, and for protecting putrefaction of vegetables and the like (B. B. McSpadden Gardener, et al., Plant Health Progress [Internet], May 10, 2002 [cited Aug. 13, 2010]. Especially, Serenade which was registered in early 2,000s by AgraQuest Co. has been produced by using B. subtilis QST713 and is registered as a fungicide and a bactericide in 25 countries, and currently, various products depending on their uses are commercially available. In addition, a research team at the USDA found that iturin antibiotics produced by B. subtilis inhibited Monilinia fructicola, the pathogen of peach brown rot, and attempted a study to develop B. subtilis as a preservative during storage of tree fruits (R. C. Gueldner, et al., J. Agric. Food Chem., 36: 366-370, 1988).
Besides, a research team lead by Dr. Pusey at the USDA found that B. subtilis has an inhibitory effect on many plant diseases (P. L. Pusey, et al., Pesticide Sci., 27: 133-140, 1989) and Phae et al. also isolated B. subtilis NB22 having a wide inhibitory effect on plant pathogens from decomposed soil for compost and proved that its active components are iturin-based materials (C. G. Phae, et al., J. Ferment. Bioeng., 69: 1-7, 1990). Like these, iturin, a cyclic peptide antibiotic, has long been widely used as a biological control agent, but, study on iturin biosynthesis genes at the molecular level has hardly been made, except for one published in J. Ferment. Bioeng. by a Japanese research team in 1990.
The complete genome sequence of B. subtilis 168 strain having a gene responsible for biosynthesis of surfactin, another cyclic peptide other than iturin was published in 1997 (Kunst, F., et al., Nature, 390: 249-256, 1997), and the result of study on a gene responsible for biosynthesis of mycosubtilin from B. subtilis ATCC 6633 was reported by a German research team in late 1990s (E. H. Duitman, et al., Proc. Natl. Acad. Sci., 96: 13294-13299, 1999). Since then, cloning, base sequence, and characteristics of iturin A gene from B. subtilis RB14 was published by a Japanese research team in 2000s (K. Tsuge, et al., J. Bacteriol., 183: 6265-6273, 2001). Furthermore, a German research team found that B. amyloliquefaciens FZB42 which promotes plant growth and suppresses plant pathogens at the same time produced cyclic peptides, surfactin, fengycin, and bacillomycin D as secondary metabolites, and investigated and reported genetic structures and functional characteristics of produced secondary metabolites at the molecular level (A. Koumoutsi, et al., J. Bacteriol., 186: 1084-1096, 2004). Besides, the above German research team reported that B. amyloliquefaciens FZB42 produces polyketide-based antibiotics, macrolactin, bacillaene, and difficidin, in addition to the above three cyclic peptides (Chen, et al., Nature Biotechnol., 25: 1007-1014, 2007). Likewise, while reports on various cyclic peptide antibiotics have been made intermittently, there is hardly a report on various kinds of iturin biosynthesis genes.
Thus, the present inventors cloned iturin biosynthesis genes from B. subtilis subsp. krictiensis ATCC 55079 (S. H. Bok, et al., U.S. Pat. No. 5,155,041, 1992. 10. 13) which is effective for various plant pathogens isolated from domestic soils and produces six kinds of iturins (iturin A to F) and obtained the U.S. patent in 1992, identified iturin biosynthesis genes by determining base sequences of the genes, analyzed their characteristics, and found that the above iturin biosynthesis genes are novel iturin biosynthesis genes that show many differences in base sequences, compared to conventional iturin genes, thereby leading to completion of the present invention.